Cloning Construction and Purification of Recombinant Mouse Foxp3 Gene

Objective To clone Mouse Foxp3 gene(MFoxp3) and construct the prokaryotic expression vector of the protein,and to express and purify mouse Foxp3 fusion protein.Methods Using RT-PCR technique,the gene encoding Mouse Foxp3 was amplified from the spleen of the mouse,and the PCR product was ligated with T Easy vector and sequenced.Then the correct clone was digested by endonuclease for releasing the fragment of mouse Foxp3,and was inserted into pET 32a(+) prokaryotic expression vector which has Trx protein with His-tag and was digested by the same endonuclease.The E.coli BL21(DE3) transformed with pET 32a(+)-Mfoxp3 were induced by IPTG for expression of MFoxp3 fusion protein.The protein was purified by Ni2+ affinity chromatography.Results The Mouse Foxp3 gene was 1,290bp and the fusion protein was expressed in BL21(DE3) at a high level by cloned into pET 32a(+).SDS-PAGE showed its molecular mass was about 63KD.Recombinant protein was purified by Ni-NTA purification column and the purity of the fusion protein was 80%.The immunogenicity and specificity were tested by Western blot assays.Conclusion The prokaryotic expression vector of mouse Foxp3 fusion protein has been constructed,and the fusion protein has been successfully expressed and purified.